Dysfunctional CD4+,CD25+ regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon-alpha-producing antigen-presenting cells

OBJECTIVE: To explore whether there are extrinsic factors that impair the suppressive function of CD4+,CD25+ regulatory T cells in patients with untreated active systemic lupus erythematosus (SLE). METHODS: We studied 15 patients with untreated active SLE, 10 patients with SLE in remission, and 15 healthy control subjects. Percentages of CD4+,CD25+,FoxP3+ Treg cells and levels of forkhead box P3 (FoxP3) protein were analyzed by flow cytometry. Expression of messenger RNA (mRNA) for FoxP3 in purified Treg cell populations was assessed by real-time polymerase chain reaction analysis. Experiments examining Treg cell function in SLE were designed to distinguish primary from secondary T cell dysfunction. Levels of interferon-alpha (IFNalpha) in supernatants from the function assays were determined with an IFN-stimulated response element-luciferase reporter assay. RESULTS: The percentage of CD4+,CD25+, FoxP3+ cells in peripheral blood was significantly increased in SLE patients as compared with controls (mean +/- SEM 9.11 +/- 0.73% versus 4.78 +/- 0.43%; P < 0.0001). We found no difference in FoxP3 expression at either the mRNA or protein level in any CD4+,CD25+ T cell subset from SLE patients as compared with controls. Antigen-presenting cells (APCs) from SLE patients were responsible for decreased Treg cell activity and could also render dysfunctional Treg cells from healthy control subjects. CD4+,CD25+ Treg cells from SLE patients exhibited normal suppressive activity when cultured with APCs from healthy controls. A partial Treg cell blockade effect was induced by the high levels of IFNalpha derived from SLE patient APCs. CONCLUSION: We suggest that blockade of Treg cell-mediated suppression by IFNalpha-producing APCs in SLE patients may contribute to a pathogenic loss of peripheral tolerance in this disease.     

Characterization of a dopamine receptor (DA2K) in the kidney inner medulla.

Dopamine (DA) produces a natriuretic/diuretic response in the kidney by mechanisms that are still not well understood. There is some indication that DA2 receptors may be involved in mediating the effects of DA, but little is known regarding the nature of this receptor in the kidney. Autoradiographic localization of [3H]spiperone, a DA2 antagonist, indicated that high-density binding was restricted to inner medullary collecting ducts (IMCDs). [3H]Spiperone binding was saturable, high affinity (Kd, 17.2 +/- 1.65 nM), and high density (Bmax, 935 +/- 83 fmol per mg of protein). The photosensitive spiperone analogue N-(p-azido-m-[125I]iodophenethyl)spiperone labeled similar sized proteins of Mr = 120,000 in membranes prepared from the kidney inner medulla, striatum, and pituitary. However, the rank-order competition profile for the [3H]spiperone binding in the kidney inner medulla differed from the DA2 receptor in striatum and pituitary and, furthermore, RNA (Northern) blot analyses of kidney inner medullary RNA with brain DA2 receptor oligonucleotide probes were negative. Functionally, DA stimulated prostaglandin E2 production by IMCD cells, an effect that could be blocked by the DA2 antagonist domperidone. These results indicate that the kidney inner medulla expresses a functional DA receptor that may represent a newly identified DA receptor subtype (here designated DA2K). Moreover, these results suggest that the kidney inner medulla may be a significant site at which DA, either directly or indirectly, influences water and electrolyte excretion.     

Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of “de novo” SCN1A Mutations in Children with Dravet Syndrome

The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling.     

Preparation,structure identification and the anti-photoaging activity of peptide fraction OP-Ia from Ostrea rivularis

The purpose of the present study is to evaluate the preparation and the structure of fraction OP-Ia and its protective effect against UV-induced photoaging through the MAPKs signaling pathway. Fractions OP-Ia and OP-Ib were prepared by enzymatic hydrolysis and purified by ultrafiltration (5 kDa) and gel chromatography (Sephadex G-25). The reducing power and superoxide radical scavenging ability were evaluated, which showed that OP-Ia had stronger antioxidant activity than OP-Ib. Next, ten peptides were identified in OP-Ia by UPLC-MS/MS. The mechanism of the anti-photoaging activity for fraction OP-Ia was investigated through the MAPKs pathway based on the HaCaT cell line. Fraction OP-Ia could inhibit the generation of ROS and the decline of cell viability induced by UV radiation, meanwhile downregulate the expression of IL-1β, IL-8, c-Jun, c-Fos, p65 NF-κB and p38 MAPK genes. Overall, the results showed that the fraction OP-Ia could be a potent component of functional foods with UV protection property.

OP-Ia could reduce ROS generation and cell viability declination induced by UV, and downregulate the expressions of IL-1β, IL-8, c-Jun, c-Fos, p65 and p38 genes.     

On the role of peptide hydrolysis for fibrillation kinetics and amyloid fibril morphology

Self-assembly of proteins into amyloid-like nanofibrils is not only a key event in several diseases, but such fibrils are also associated with intriguing biological function and constitute promising components for new biobased materials. The bovine whey protein β-lactoglobulin has emerged as an important model protein for the development of such materials. We here report that peptide hydrolysis is the rate-determining step for fibrillation of β-lactoglobulin in whey protein isolate. We also explore the observation that β-lactoglobulin nanofibrils of distinct morphologies are obtained by simply changing the initial protein concentration. We find that the morphological switch is related to different nucleation mechanisms and that the two classes of nanofibrils are associated with variations of the peptide building blocks. Based on the results, we propose that the balance between protein concentration and the hydrolysis rate determines the structure of the formed nanofibrils.

Peptide hydrolysis determines the fibrillation rate and the morphology of amyloid-like nanofibrils formed by β-lactoglobulin at low pH.     

Effects of multiwalled carbon nanotubes on CH4 hydrate in the presence of tetra-n-butyl ammonium bromide

Hydrate formation is an important technology for gas storage and transportation. In this work, the effect of multiwalled carbon nanotubes (MWCNTs) on CH₄ hydrate formation was examined by determining the phase equilibrium conditions and kinetics characteristics of a mixed system of CH₄, tetra-n-butyl ammonium bromide (TBAB), and MWCNTs. The phase equilibrium was examined in the temperature range of 286.13–293.04 K and the pressure range of 0.55–6.56 MPa for various mass fractions of MWCNTs (0.004, 0.1, 0.5, and 1.0 wt%). In the CH₄ + TBAB system, the presence of MWCNTs was found to shift the phase equilibrium conditions to a lower temperature by about 1 K compared with those in the absence of MWCNTs. However, the concentration of MWCNTs had little effect on the phase equilibrium conditions. When the concentration of MWCNTs was 1.0 wt%, the addition of MWCNTs reduced the induction time of hydrate formation by 79.5%. When the concentration of MWCNTs was 0.1 wt%, the addition of MWCNTs enhanced the hydrate growth rate by 61.5%. Powder X-ray diffraction patterns revealed that hydrates with orthorhombic structures (corresponding to TBAB·38H₂O with 3D cages) were formed in the systems with and without MWCNTs. Moreover, peaks corresponding to MWCNTs were not observed in the patterns of the hydrates and the addition of MWCNTs had no influence on the structure and type of hydrate. Thus, MWCNTs were not incorporated into the hydrate cages.

Hydrate formation is an important technology for gas storage and transportation.     

Hybrid scaffolds of Mg alloy mesh reinforced polymer/extracellular matrix composite for critical‐sized calvarial defect reconstruction

The challenge of developing scaffolds to reconstruct critical‐sized calvarial defects without the addition of high levels of exogenous growth factor remains relevant. Both osteogenic regenerative efficacy and suitable mechanical properties for the temporary scaffold system are of importance. In this study, a Mg alloy mesh reinforced polymer/demineralized bone matrix (DBM) hybrid scaffold was designed where the hybrid scaffold was fabricated by a concurrent electrospinning/electrospraying of poly(lactic‐co‐glycolic acid) (PLGA) polymer and DBM suspended in hyaluronic acid (HA). The Mg alloy mesh significantly increased the flexural strength and modulus of PLGA/DBM hybrid scaffold. In vitro results demonstrated that the Mg alloy mesh reinforced PLGA/DBM hybrid scaffold (Mg‐PLGA@HA&DBM) exhibited a stronger ability to promote the proliferation of bone marrow stem cells (BMSCs) and induce BMSC osteogenic differentiation compared with control scaffolding materials lacking critical components. In vivo osteogenesis studies were performed in a rat critical‐sized calvarial defect model and incorporated a variety of histological stains and immunohistochemical staining of osteocalcin. At 12 weeks, the rat model data showed that the degree of bone repair for the Mg‐PLGA@HA&DBM scaffold was significantly greater than for those scaffolds lacking one or more of the principal components. Although complete defect filling was not achieved, the improved mechanical properties, promotion of BMSC proliferation and induction of BMSC osteogenic differentiation, and improved promotion of bone repair in the rat critical‐sized calvarial defect model make Mg alloy mesh reinforced PLGA/DBM hybrid scaffold an attractive option for the repair of critical‐sized bone defects where the addition of exogenous isolated growth factors is not employed.     

Using biomimetically mineralized collagen membranes with different surface stiffness to guide regeneration of bone defects

Because guided bone regeneration (GBR) process is pronouncedly affected by the micro‐environment in the defect, the surface stiffness of collagen membranes as a constituent part of the micro‐environment was investigated in this study. The objective of this study was to manufacture biomimetically mineralized collagen membranes with controllable surface stiffness based on biomimetic strategy and to investigate the influences of surface stiffness on GBR process. The characterization and biocompatibility of membranes were examined in vitro. The mechanical properties of membranes were evaluated on macro and micro levels using tensile test and atomic force microscope, respectively. The critical‐size cranial defect model and ectopic osteogenesis were chosen to employ their performances in vivo. The results indicated that the biomimetically mineralized collagen membranes with controllable surface stiffness were manufactured based on the biomimetic theory. The in vitro experiments showed that the mineralized collagen membrane with satisfactory surface stiffness can better promote the adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells. The membranes can perform excellently in both osteoinduction and osteoconduction, which results in effective manifestations in aspects of ectopic osteogenesis and GBR in vivo. Therefore, this biomimetically mineralized collagen membrane is a promising candidate for GBR treatment in future.